Streptococcus thermophilus is a Gram-positive thermophilic bacterium used globally as a starter culture in dairy fermentations and is widely employed for the production of cheese and yoghurt products. Despite its usefulness in starter cultures, S. thermophilus remains highly susceptible to (bacterio)phage predation which can lead to substandard or failed fermentations and considerable economic losses. Evidenced by these potentially considerable costs, there is a clear advantage to selecting robust starters which are less susceptible to phage attack and yet retain favourable growth and production characteristics. Combined with effective hygiene and sanitation in industrial fermentation plants, unrelated robust starters used in rotation have the potential to reduce the incidence of phage fermentation disruption.
Phages of S. thermophilus are, despite their narrow host ranges, the major cause of fermentation failure, due to their short latent period and large burst sizes. They are generally classified as Siphoviridae (having isometric heads and long, non-contractile tails) and usually fall into two groups (cos- and pac-type), based on their mode of DNA packaging and the number of major structural proteins present (Le Marrec et al., 1997. Applied and Environmental Microbiology 63 (8), p. 3246-3253—Two groups of bacteriophages infecting Streptococcus thermophilus can be distinguished on the basis of mode of packaging and genetic determinants for major structural proteins). More recently, a third group of phages infecting S. thermophilus was identified that represents a novel genetic lineage and highlights the genetic plasticity of these phages (Mills et al., 2011. International Dairy Journal 21, p. 963-969—A new phage on the ‘Mozzarella’ block: Bacteriophage 5093 shares a low level of homology with other Streptococcus thermophilus phages). Consequently, phages of S. thermophilus persist in dairy fermentation facilities leading to starter culture infections. In reponse to these infections, microorganisms such as S. thermophilus has evolved several mechanisms of phage resistance, some of which are more effective and stable than others.
Mutants which have become resistant to phages by means of effective and stable mechanisms may be characterised by means of DNA sequencing, morphological analyses and/or adsorption assays.
Bacteriophage resistance systems have evolved in microorganisms such as S. thermophilus in tandem with phage adaptation strategies to overcome these biological barriers. These systems can include those preventing phage adsorption, blocking DNA injection, restriction/modification of DNA (R/M) and abortive infection or Abi (Labrie et al. (2010) Nature reviews 8, p. 317-327—Bacteriophage resistance mechanisms). To date, the most intensely characterised and the most frequent of these systems in lactic streptococci, are the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, which are known to provide acquired immunity to phages through an RNA-mediated dsDNA targeting process (Barrangou et al. (2007). Science 315, p. 1709-1712—CRISPR provides acquired resistance against viruses in prokaryotes; Garneau et al. (2010). Nature 468, p. 67-71—The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA).
Three distinct CRISPR systems (CRISPRs 1, 2 and 3), representing two distinct types (types II and III) are widespread in S. thermophilus and individual strains may contain multiple systems. Diversity was observed across three CRISPR loci between 124 different S. thermophilus strains. Specifically, CRISPR1 was ubiquitous, whereas CRISPR2 was present in 59 of 65 strains, and CRISPR3 was present in 53 of 66 strains. A total of 49 strains (39.5%) carried all three loci. (Horvath et al., 2008. Journal of Bacteriology 190 (4), p. 1401-1412—Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus). Recently, a fourth CRISPR system has been described (Sinkunas et al., 2013. The European Molecular Biology Organisation journal 32, p. 385-394—In vitro reconstitution of cascade-mediated CRISPR immunity in Streptococcus thermophilus) although its prevalence is rare and in vivo activity is not known. Although CRISPR provides effective immunity against phages through acquired spacers which are identical to short regions of the attacking phage genomes (Barrangou et al., 2007, as above), it is known that phages can rapidly evolve to overcome these spacer additions through single nucleotide alterations in the corresponding genomic region (Deveau et al., 2008. Journal of Bacteriology 190 (4), p. 1390-1400—Phage response to CRISPR-encoded resistance in Streptococcus thermophilus). Furthermore, since CRISPR mutations are the most frequent mutations involved in phage resistance it is difficult to identify other more desirable mutations which provide phage resistance. Therefore, it is desirable to develop a method to obtain phage-resistant derivatives of microorganism parent strains suitable for food and feed fermentation, and especially S. thermophilus, where such phage resistance is due to the action of alternative phage resistance mechanisms than CRISPR. The present invention provides a method to construct and select for such phage-resistant bacteria.